The present invention relates to an enzyme electrode or electrochemical biosensor, which may be used for electrochemically measuring the concentration of and/or monitoring the level of one or more selected components in a liquid mixture or liquid environment.
These electrochemical biosensors may be used in the chemical, food and biochemical industries and in clinical applications in animal or human medicine, in particular to measure in vivo the concentration of components in body fluids.
These electrochemical biosensors will be described with reference to one particular measurement, the determination of the glucose concentration in an aqueous mixture. While the measurement of glucose concentration is one object of the invention, other and broader objects are not hereby excluded.
Typical amperometric glucose electrodes based on glucose oxidase (GO) undergo several chemical or electrochemical steps which produce a measurable current which is linearly related to the glucose concentration. In the initial step, glucose converts the oxidized flavin adenine dinucleotide (FAD) center of the enzyme into its reduced form (FADH.sub.2). Because these redox centers are located well within the enzyme molecule, direct electron transfer to the surface of a conventional electrode does not occur to any measurable degree. A common method of indirectly measuring the amount of reduced glucose oxidase, and hence the amount of glucose present, relies on the natural enzymatic reaction as described in Biosensors: Fundamentals and Applications (Oxford University Press, New York, 1987) Chapter 1, and shown by the following reaction formula: ##STR1## In this reaction, oxygen is the electron acceptor for glucose oxidase. Glucose is oxidized (dehydrogenated) to gluconolactone through the catalytic reaction caused by glucose oxidase, while oxygen is reduced to H.sub.2 O.sub.2. The concentration of O.sub.2 and H.sub.2 O.sub.2 can be measured with conventional electrochemical techniques, whereby it is possible to obtain the concentration of glucose indirectly by means of the measurement of O.sub.2 consumption as well as H.sub.2 O.sub.2 formation governed by the reaction depicted above. In this measuring scheme the sensor has the disadvantage of being sensitive to the concentration of O.sub.2.
Instead of the natural electron acceptor O.sub.2, an artificial electron acceptor (mediator) may be used to shuttle electrons between the reduced flavin adenine dinucleotide and the electrode by the following mechanism, described in Anal. Chem. 56, 667-671 (1984): ##STR2## The preferred mediating species M may be, but is not limited to, ferrocene or a substituted ferrocene.
U.S. Pat. Nos. 4,545,382 and 4,711,245 disclose an enzyme electrode, in which the mediator is a ferrocene or substituted ferrocene molecule. In potential clinical applications, or where long term stability is desirable, sensors based on electron-shuttling redox couples suffer from an inherent drawback: the soluble, or partially soluble, mediating species can diffuse away from the electrode surface into the bulk of the solution. This precludes the use of these devices in implantable probes.
U.S. Pat. No. 4,224,125 discloses an enzyme electrode, in which the water soluble mediator is in polymeric form in order to remain immobilized near the electrode surface by being too large to diffuse through a retaining membrane into the bulk of the solution. The polymeric redox mediator is reduced by the enzyme catalytic process and reoxidized by the electrode, in the vicinity of which it is contained. This electrode design requires a retaining membrane which is a disadvantage for microelectrode applications or where rapid response and high sensitivity are important features of the electrode.
It is desirable to find a mediator which can rapidly transfer electrons between the enzyme and the electrode at a rate corresponding to the rate of the enzyme-catalyzed reaction.
It is further desirable to use a mediating species which is covalently attached in such a fashion as to make it insoluble in the solution to be analyzed, thus preventing the mediating species from diffusing away from the electrode surface.
It is specifically desirable to find a mediator which is relatively insensitive to the presence of interfering substances, in particular oxygen.
These objects are accomplished by the present invention wherein the mediating species is chemically bound to a flexible polymer backbone which allows close contact between the FAD/FADH.sub.2 centers of the enzyme and the attached mediator, yet prevents the latter from diffusing away from the electrode surface.
The present invention covers a class of redox polymers which has exceptional properties for mediating enzyme-catalyzed reactions in electrode sensing systems. The redox polymer acts as an electron transfer relay system in a manner similar to that described by Degani and Heller [(J. Phys. Chem. 91, 1285-1289 (1987)] where the electron relays are covalently attached to the enzyme itself. A disadvantage of this prior art design is measurably reduced enzyme activity. A further disadvantage is that the applicability of this design is limited to enzymes which allow this particular attachment chemistry. In the present invention, the necessary electrical communication between the FAD/FADH.sub.2 centers and the electrode is achieved without modifying the enzyme. A key aspect of the present invention is the use of a highly flexible polymer backbone with sufficient local conformational mobility to allow the attached mediator species to come in close proximity to the enzyme catalytic center, thereby acting as an efficient electron transfer relay to an electron collector. The present system is applicable to all oxido-reductase enzymes, including enzymes which have been modified according to the scheme of Degani and Heller.
One aspect of the invention is to provide a network of donor/acceptor relays covalently attached to a flexible polymer backbone. In another aspect of the invention the flexible polymer backbone is provided by a siloxane polymer. The unique flexibility of the polysiloxane backbone, which has virtually no energy barrier to rotation, allows these relay moieties to interact intimately with the enzyme molecule and achieve a close contact with the FAD/FADH.sub.2 centers. This is a vital consideration since, as has been demonstrated [J. Electroanal. Chem. 250, 417-425 (1988)], less flexible redox polymers such as poly(vinylferrocene) cannot achieve a sufficiently close contact with the enzyme's redox centers to serve as effective electron transfer relay systems.
An object of the invention is to provide an enzyme electrode for use in liquid mixtures of components for detecting the presence of, measuring the amount of and/or monitoring the level of one or more selected components capable of undergoing an enzyme-catalyzed reaction, in which an oxido-reductase enzyme as well as a polymeric mediator system which transfers electrons to an electron collector when the enzyme is catalytically active, are both maintained in an immobilized state on at least an external surface of the electron collector.
A further object of the invention is to provide an enzyme electrode of the above described type in which the polymeric mediator is comprised of a flexible polymer backbone onto which is covalently attached molecular mediator compounds such as to form a donor-acceptor electron relay system.
A further object of the invention is to provide a polymeric mediator compound which is insoluble in aqueous mixtures such as to remain immobilized on the electrode surface without a retaining membrane.
A further object of the present invention is to provide an enzyme electrode of the above described type which can be formed on a small scale to be used for in vivo concentration measurements.